Skip to main content

Publications search

Found 34428 matches. Displaying 11-20
Boyaci H, Chen J, Jansen R, Darst SA, Campbell EA
Show All Authors

Structures of an RNA polymerase promoter melting intermediate elucidate DNA unwinding

NATURE 2019 JAN 17; 565(7739):382-385
A key regulated step of transcription is promoter melting by RNA polymerase (RNAP) to form the open promoter complex(1-3). To generate the open complex, the conserved catalytic core of the RNAP combines with initiation factors to locate promoter DNA, unwind 12-14 base pairs of the DNA duplex and load the template-strand DNA into the RNAP active site. Formation of the open complex is a multi-step process during which transient intermediates of unknown structure are formed(4-6). Here we present cryo-electron microscopy structures of bacterial RNAP-promoter DNA complexes, including structures of partially melted intermediates. The structures show that late steps of promoter melting occur within the RNAP cleft, delineate key roles for fork-loop 2 and switch 2-universal structural features of RNAP-in restricting access of DNA to the RNAP active site, and explain why clamp opening is required to allow entry of single-stranded template DNA into the active site. The key roles of fork-loop 2 and switch 2 suggest a common mechanism for late steps in promoter DNA opening to enable gene expression across all domains of life.
Li C, Bonazzoli E, Bellone S, Choi J, Dong WL, Menderes G, Altwerger G, Han C, Manzano A, Bianchi A, Pettinella F, Manara P, Lopez S, Yadav G, Riccio F, Zammataro L, Zeybek B, Yang-Hartwich Y, Buza N, Hui P, Wong S, Ravaggi A, Bignotti E, Romani C, Todeschini P, Zanotti L, Zizioli V, Odicino F, Pecorelli S, Ardighieri L, Silasi DA, Litkouhi B, Ratner E, Azodi M, Huang GS, Schwartz PE, Lifton RP, Schlessinger J, Santin AD
Show All Authors

Mutational landscape of primary, metastatic, and recurrent ovarian cancer reveals c-MYC gains as potential target for BET inhibitors

Ovarian cancer remains the most lethal gynecologic malignancy. We analyzed the mutational landscape of 64 primary, 41 metastatic, and 17 recurrent fresh-frozen tumors from 77 patients along with matched normal DNA, by whole-exome sequencing (WES). We also sequenced 13 pairs of synchronous bilateral ovarian cancer (SBOC) to evaluate the evolutionary history. Lastly, to search for therapeutic targets, we evaluated the activity of the Bromodomain and Extra-Terminal motif (BET) inhibitor GS-626510 on primary tumors and xenografts harboring c-MYC amplifications. In line with previous studies, the large majority of germline and somatic mutations were found in BRCA1/2 (21%) and TP53 (86%) genes, respectively. Among mutations in known cancer driver genes, 77% were transmitted from primary tumors to metastatic tumors, and 80% from primary to recurrent tumors, indicating that driver mutations are commonly retained during ovarian cancer evolution. Importantly, the number, mutation spectra, and signatures in matched primary-metastatic tumors were extremely similar, suggesting transcoelomic metastases as an early dissemination process using preexisting metastatic ability rather than an evolution model. Similarly, comparison of SBOC showed extensive sharing of somatic mutations, unequivocally indicating a common ancestry in all cases. Among the 17 patients with matched tumors, four patients gained PIK3CA amplifications and two patients gained c-MYC amplifications in the recurrent tumors, with no loss of amplification or gain of deletions. Primary cell lines and xenografts derived from chemotherapy-resistant tumors demonstrated sensitivity to JQ1 and GS-626510 (P = 0.01), suggesting that oral BET inhibitors represent a class of personalized therapeutics in patients harboring recurrent/chemotherapy-resistant disease.
Lorenzen E, Sakmar TP
Show All Authors

Receptor Structures for a Caldron of Cannabinoids

CELL 2019 JAN 24; 176(3):409-411
Structures of the cannabinoid receptor 1 (CB1) in complex with an "ultrapotent'' synthetic cannabinoid and its G protein (Krishna Kumar et al., 2019) and CB2 in complex with a new rationally designed inverse agonist (Li et al., 2019) provide unique snapshots of the molecular pharmacology of cannabinoids.
Galea S, Vaughan R
Show All Authors

Creating Policy-Relevant Evidence for Population Health Science: A Public Health of Consequence, January 2019

Hatzi K, Geng HM, Doane AS, Meydan C, LaRiviere R, Cardenas M, Duy C, Shen H, Vidal MNC, Baslan T, Mohammad HP, Kruger RG, Shaknovich R, Haberman AM, Inghirami G, Lowe SW, Melnick AM
Show All Authors

Histone demethylase LSD1 is required for germinal center formation and BCL6-driven lymphomagenesis

NATURE IMMUNOLOGY 2019 JAN; 20(1):86-96
Germinal center (GC) B cells feature repression of many gene enhancers to establish their characteristic transcriptome. Here we show that conditional deletion of Lsd1 in GCs significantly impaired GC formation, associated with failure to repress immune synapse genes linked to GC exit, which are also direct targets of the transcriptional repressor BCL6. We found that BCL6 directly binds LSD1 and recruits it primarily to intergenic and intronic enhancers. Conditional deletion of Lsd1 suppressed GC hyperplasia caused by constitutive expression of BCL6 and significantly delayed BCL6-driven lymphomagenesis. Administration of catalytic inhibitors of LSD1 had little effect on GC formation or GC-derived lymphoma cells. Using a CRISPR-Cas9 domain screen, we found instead that the LSD1 Tower domain was critical for dependence on LSD1 in GC-derived B cells. These results indicate an essential role for LSD1 in the humoral immune response, where it modulates enhancer function by forming repression complexes with BCL6.
Waters EM, Mazid S, Dodos M, Puri R, Janssen WG, Morrison JH, McEwen BS, Milner TA
Show All Authors

Effects of estrogen and aging on synaptic morphology and distribution of phosphorylated Tyr1472 NR2B in the female rat hippocampus

NEUROBIOLOGY OF AGING 2019 JAN; 73(?):200-210
Age and estrogens may impact the mobility of N-methyl-D-aspartate receptors (NMDARs) in hippocampal synapses. Here, we used serial section immunogold electron microscopy to examine whether phosphorylated tyrosine 1472 NR2B (pY1472), which is involved in the surface expression of NMDARs, is altered in the dorsal hippocampus of young (3-4 months old) and aged ( similar to 24 months old) ovariectomized rats treated with 17 beta-estradiol or vehicle for 2 days. The number of gold particles labeling pY1472 was higher in presynaptic and postsynaptic compartments of aged rats with low estradiol (vehicle-treated) compared to other groups. In terminals, pY1472 levels were elevated in aged rats but reduced by estradiol treatment to levels seen in young rats. Conversely, the mitochondria number was lower in aged females but was restored to young levels by estradiol. In the postsynaptic density and dendritic spines, estradiol reduced pY1472 in young and aged rats. As phosphorylation at Y1472 blocks NR2B endocytosis, reduction of pY1472 by estradiol suggests another mechanism through which estrogen enhances synaptic plasticity by altering localization of NMDAR subunits within synapses. (C) 2018 Elsevier Inc. All rights reserved.
Mayle R, Langston L, Molloy KR, Zhang D, Chait BT, O'Donnell ME
Show All Authors

Mcm10 has potent strand-annealing activity and limits translocase-mediated fork regression

The 11-subunit eukaryotic replicative helicase CMG (Cdc45, Mcm2-7, GINS) tightly binds Mcm10, an essential replication protein in all eukaryotes. Here we show that Mcm10 has a potent strand-annealing activity both alone and in complex with CMG. CMG-Mcm10 unwinds and then reanneals single strands soon after they have been unwound in vitro. Given the DNA damage and replisome instability associated with loss of Mcm10 function, we examined the effect of Mcm10 on fork regression. Fork regression requires the unwinding and pairing of newly synthesized strands, performed by a specialized class of ATP-dependent DNA translocases. We show here that Mcm10 inhibits fork regression by the well-known fork reversal enzyme SMARCAL1. We propose that Mcm10 inhibits the unwinding of nascent strands to prevent fork regression at normal unperturbed replication forks, either by binding the fork junction to form a block to SMARCAL1 or by reannealing unwound nascent strands to their parental template. Analysis of the CMG-Mcm10 complex by cross-linking mass spectrometry reveals Mcm10 interacts with six CMG subunits, with the DNA-binding region of Mcm10 on the N-face of CMG. This position on CMG places Mcm10 at the fork junction, consistent with a role in regulating fork regression.
Choi YJ, Halbritter J, Braun DA, Scheeler M, Schapiro D, Rim JH, Nandadasa S, Choi WI, Widmeier E, Shril S, Korber F, Sethi SK, Lifton RP, Beck BB, Apte SS, Gee HY, Hildebrandt F
Show All Authors

Mutations of ADAMTS9 Cause Nephronophthisis-Related Ciliopathy

Nephronophthisis-related ciliopathies (NPHP-RCs) are a group of inherited diseases that are associated with defects in primary cilium structure and function. To identify genes mutated in NPHP-RC, we performed homozygosity mapping and whole-exome sequencing for >100 individuals, some of whom were single affected individuals born to consanguineous parents and some of whom were siblings of indexes who were also affected by NPHP-RC. We then performed high-throughput exon sequencing in a worldwide cohort of 800 additional families affected by NPHP-RC. We identified two ADAMTS9 mutations (c.4575_4576de1 [p.Gln1525Hisfs*60] and c.194C>G [p.Thr65Arg]) that appear to cause NPHP-RC. Although ADAMTS9 is known to be a secreted extracellular metalloproteinase, we found that ADAMTS9 localized near the basal bodies of primary cilia in the cytoplasm. Heterologously expressed wild-type ADAMTS9, in contrast to mutant proteins detected in individuals with NPHP-RC, localized to the vicinity of the basal body. Loss of ADAMTS9 resulted in shortened cilia and defective sonic hedgehog signaling. Knockout of Adamts9 in IMCD3 cells, followed by spheroid induction, resulted in defective lumen formation, which was rescued by an overexpression of wild-type, but not of mutant, ADAMTS9. Knockdown of adamts9 in zebrafish recapitulated NPHP-RC phenotypes, including renal cysts and hydrocephalus. These findings suggest that the identified mutations in ADAMTS9 cause NPHP-RC and that ADAMTS9 is required for the formation and function of primary cilia.
Nagy Z, Vogtle T, Geer MJ, Mori J, Heising S, Di Nunzio G, Gareus R, Tarakhovsky A, Weiss A, Neel BG, Desanti GE, Mazharian A, Senis YA
Show All Authors

The Gp1ba-Cre transgenic mouse: a new model to delineate platelet and leukocyte functions

BLOOD 2019 JAN 24; 133(4):331-343
Conditional knockout (KO) mouse models are invaluable for elucidating the physiological roles of platelets. The Platelet factor 4-Cre recombinase (Pf4-Cre) transgenic mouse is the current model of choice for generating megakaryocyte/platelet-specific KO mice. Platelets and leukocytes work closely together in a wide range of disease settings, yet the specific contribution of platelets to these processes remains unclear. This is partially a result of the Pf4-Cre transgene being expressed in a variety of leukocyte populations. To overcome this issue, we developed a Gp1ba-Cre transgenic mouse strain in which Cre expression is driven by the endogenous Gp1ba locus. By crossing Gp1ba-Cre and Pf4-Cre mice to the mT/mG dual-fluorescence reporter mouse and performing a head-to-head comparison, we demonstrate more stringent megakaryocyte lineage-specific expression of the Gp1ba-Cre transgene. Broader tissue expression was observed with the Pf4-Cre transgene, leading to recombination in many hematopoietic lineages, including monocytes, macrophages, granulocytes, and dendritic and B and T cells. Direct comparison of phenotypes of Csk, Shp1, or CD148 conditional KO mice generated using either the Gp1ba-Cre or Pf4-Cre strains revealed similar platelet phenotypes. However, additional inflammatory and immunological anomalies were observed in Pf4-Cre-generated KO mice as a result of nonspecific deletion in other hematopoietic lineages. By excluding leukocyte contributions to phenotypes, the Gp1ba-Cre mousewill advance our understanding of the role of platelets in inflammation and other pathophysiological processes in which platelet-leukocyte interactions are involved.
Nasca C, Rasgon N, McEwen B
Show All Authors

An emerging epigenetic framework of systemic and central mechanisms underlying stress-related disorders